RESUMEN
In the present study, the distribution of sulphated glycosaminoglycans (GAGs) in the developing vertebral column of Atlantic salmon (Salmo salar) at 700, 900, 1100 and 1400 d° was examined by light microscopy. The mineralization pattern was outlined by Alizarin red S and soft structures by Alcian blue. The temporal and spatial distribution patterns of different types of GAGs: chondroitin-4-sulphate/dermatan sulphate, chondroitin-6-sulphate, chondroitin-0-sulphate and keratan sulphate were addressed by immunohistochemistry using monoclonal antibodies against the different GAGs. The specific pattern obtained with the different antibodies suggests a unique role of the different GAG types in pattern formation and mineralization. In addition, the distribution of the different GAG types in normal and malformed vertebral columns from 15 g salmon was compared. A changed expression pattern of GAGs was found in the malformed vertebrae, indicating the involvement of these molecules during the pathogenesis. The molecular size of proteoglycans (PGs) in the vertebrae carrying GAGs was analysed with western blotting, and mRNA transcription of the PGs aggrecan, decorin, biglycan, fibromodulin and lumican by real-time qPCR. Our study reveals the importance of GAGs in development of vertebral column also in Atlantic salmon and indicates that a more comprehensive approach is necessary to completely understand the processes involved.
Asunto(s)
Glicosaminoglicanos/metabolismo , Notocorda/metabolismo , Proteoglicanos/metabolismo , Salmo salar/metabolismo , Columna Vertebral/metabolismo , Animales , Notocorda/anomalías , Notocorda/anatomía & histología , Salmo salar/anomalías , Salmo salar/anatomía & histología , Columna Vertebral/anomalías , Columna Vertebral/anatomía & histologíaRESUMEN
We analysed the distribution and expression of the small leucine-rich proteoglycans (SLRPs) decorin, biglycan and lumican in vertebral columns of Atlantic salmon Salmo salar L. with and without radiographically detectable deformities. Vertebral deformities are a reoccurring problem in salmon and other intensively farmed species, and an understanding of the components involved in the pathologic development of the vertebrae is important in order to find adequate solutions to this problem. Using immunohistology and light microscopy, we found that in non-deformed vertebrae biglycan, lumican and decorin were all expressed in osteoblasts at the vertebral growth zones and at the ossification front of the chondrocytic arches. Hence, the SLRPs are expressed in regions where intramembranous and endochondral ossification take place. In addition, mRNA expression of biglycan, decorin and lumican was demonstrated in a primary osteoblast culture established from Atlantic salmon, supporting the in vivo findings. Transcription of the SLRPs increased during differentiation of the osteoblasts in vitro and where lumican mRNA expression increased later in the differentiation compared with decorin and biglycan. Intriguingly, in vertebral fusions, biglycan, decorin and lumican protein expression was extended to trans-differentiating cells at the border between arch centra and osteoblast growth zones. In addition, mRNA expression of biglycan, decorin and lumican differed between non-deformed and fused vertebrae, as shown by quantitative PCR (qPCR). Western blotting revealed an additional band of biglycan in fused vertebrae which had a higher molecular weight than in non-deformed vertebrae. Fourier-transform infrared (FTIR) spectroscopy revealed more spectral focality in the endplates of vertebral fusions and significantly more non-reducible collagen crosslinks compared with non-deformed vertebrae, thus identifying differences in bone structure.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteoglicanos/metabolismo , ARN Mensajero/metabolismo , Salmo salar/anatomía & histología , Salmo salar/metabolismo , Columna Vertebral/anatomía & histología , Animales , Proteoglicanos/química , Proteoglicanos/genética , ARN Mensajero/genética , TiempoRESUMEN
Knowledge on fish matrix biology is important to ensure optimal fish -quality, -growth and -health in aquaculture. The aquaculture industry face major challenges related to matrix biology, such as inflammations and malformations. Atlantic cod skeletal muscle was investigated for collagen I, decorin, biglycan, and lumican expression and distribution by real-time PCR, immunohistochemical staining and Western blotting. Immunohistochemical staining and Western immunoblotting were also performed using antibodies against glycosaminoglycan side chains of these proteoglycans, in addition to fibromodulin. Real-time PCR showed highest mRNA expression of lumican and collagen I. Collagen I and proteoglycan immunohistochemical staining revealed distinct thread-like structures in the myocommata, with the exception of fibromodulin, which stained in dense structures embedded in the myocommata. Chondroitinase AC-generated epitopes stained more limited than cABC-generated epitopes, indicating a stronger presence of dermatan sulfate than chondroitin sulfate in cod muscle. Lumican and keratan sulfate distribution patterns were strong and ubiquitous in endomysia and myocommata. Western blots revealed similar SLRPs sizes in cod as are known from mammals. Staining of chondroitin/dermatan sulfate epitopes in Western blots were similar in molecular size to those of decorin and biglycan, whereas staining of keratan sulfate epitopes coincided with expected molecular sizes of lumican and fibromodulin. In conclusion, lumican was a major proteoglycan in cod muscle with ubiquitous distribution overlapping with keratan sulfate. Other leucine-rich proteoglycans were also present in cod muscle, and Western blot using antibodies developed for mammalian species showed cross reactivity with fish, demonstrating similar structures and molecular weights as in mammals.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/análisis , Matriz Extracelular/química , Gadus morhua/metabolismo , Sulfato de Queratano/análisis , Músculo Esquelético/química , Animales , Biglicano/análisis , Western Blotting , Colágeno Tipo I/análisis , Decorina/análisis , Proteínas de la Matriz Extracelular/análisis , Fibromodulina , Leucina/análisis , Lumican , Proteoglicanos/análisis , ARN Mensajero/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Coloración y EtiquetadoRESUMEN
We have previously characterized the development of vertebral fusions induced by elevated water temperature in Atlantic salmon. Molecular markers of bone and cartilage development together with histology were used to understand the complex pathology and mechanism in the development of this spinal malformation. In this study, we wanted to use proteomics, a non-hypothetical approach to screen for possible new markers involved in the fusion process. Proteins extracted from non-deformed and fused vertebrae of Atlantic salmon were therefore compared by two-dimensional electrophoresis (2DE) and MALDI-TOF analysis. Data analysis of protein spots in the 2DE gels demonstrated matrilin-1, also named cartilage matrix protein, to be the most highly up-regulated protein in fused compared with non-deformed vertebrae. Furthermore, real-time PCR analysis showed strong up-regulation of matrilin-1 mRNA in fused vertebrae. Immunohistochemistry demonstrated induced matrilin-1 expression in trans-differentiating cells undergoing a metaplastic shift toward chondrocytes in fusing vertebrae, whereas abundant expression was demonstrated in cartilaginous tissue and chordocytes of both non-deformed and fused vertebrae. These results identifies matrilin-1 as a new interesting candidate in the fusion process, and ratify the use of proteomic as a valuable technique to screen for markers involved in vertebral pathogenesis.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Deformidades Adquiridas de la Articulación/metabolismo , Salmo salar/metabolismo , Columna Vertebral/metabolismo , Animales , Biomarcadores/metabolismo , Transdiferenciación Celular , Electroforesis en Gel Bidimensional , Proteínas de Peces/metabolismo , Proteínas Matrilinas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Columna Vertebral/patologíaRESUMEN
The presence of sulfated glycosaminoglycans (GAGs) was demonstrated in the connective tissue of bovine and cod skeletal muscle by histochemical staining using Alcian blue added MgCl(2) (0.06 M and 0.4 M, respectively). For further identification of the sulfated GAGs, a panel of monoclonal antibodies, 1B5, 2B6, 3B3 and 5D4 was used that recognizes epitopes in chondroitin-0-sulfate (C0S), chondroitin-4-sulfate/dermatan sulfate (C4S/DS), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), respectively. Light microscopy and Western blotting techniques showed that in bovine and cod muscle C0S and C6S were primarily localized pericellularly, whereas cod exhibited a more intermittent staining. C4S was expressed around the separate cells and also in the perimysium and myocommata. In contrast to bovine muscle, which hardly expressed highly sulfated KS, cod exhibited a very strong and consistent staining. Western blotting showed that C0S and C6S were mainly associated with proteoglycans (PGs) of high molecular sizes in both species. Contrary to bovine muscle, C4S in cod was associated with molecules of various sizes. Both cod and bovine muscle contained KSPGs of similar sizes as C4S. KSPGs of different sizes and buoyant densities, sensitive to keratanase I and II were found expressed in cod.
Asunto(s)
Tejido Conectivo/metabolismo , Gadus morhua/metabolismo , Glicosaminoglicanos/inmunología , Glicosaminoglicanos/metabolismo , Músculo Esquelético/metabolismo , Animales , Anticuerpos Monoclonales , Western Blotting , Bovinos , Electroforesis en Gel de Poliacrilamida , Epítopos/inmunología , MasculinoRESUMEN
Heparan sulfate proteoglycans (HSPGs) were identified in pre-rigor muscle of two species of cold water fish, Atlantic cod (Gadus morhua) and spotted wolffish (Anarhichas minor) by biochemical and immunological methods. The distribution was described by immunohistology. Special emphasis was directed to the extracellular matrix (ECM) HSPGs perlecan and agrin. In vivo 35S-sulfate labeling combined with ultracentrifugation in CsCl2, DEAE chromatography and scintillation counting of the eluates, revealed that the content of 35S-labeled PGs was much higher in wolffish than in cod. A considerable proportion of the 35S-sulfated PGs in both species was HSPG, as judged by nitrous acid degradation. HSPG represented, however, a higher proportion of the 35S-sulfated PGs in cod compared to wolffish. Dot blot and electrophoresis/western blot using two different HS-mAbs, 10E4 and HepSS-1 indicated structural differences in the HS-chains of the PGs present. This observation was strengthened by immunohistochemistry, showing that both mAbs detected epitopes in the pericellular area, but the staining patterns were not superimposable. Two different agrin isoforms were identified in both species. Furthermore, in the white muscle of both cod and wolffish, perlecan mAb (A7L6) showed positive staining restricted to the transition between myocommata and myofibers.
Asunto(s)
Agrina/química , Matriz Extracelular/química , Proteínas de Peces/química , Gadus morhua , Proteoglicanos de Heparán Sulfato/química , Músculo Esquelético/química , Agrina/aislamiento & purificación , Agrina/metabolismo , Matriz Extracelular/metabolismo , Proteínas de Peces/aislamiento & purificación , Proteínas de Peces/metabolismo , Gadus morhua/metabolismo , Proteoglicanos de Heparán Sulfato/aislamiento & purificación , Proteoglicanos de Heparán Sulfato/metabolismo , Músculo Esquelético/metabolismo , Miofibrillas/química , Miofibrillas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Especificidad de la EspecieRESUMEN
Two species of commercially important cold water fish were investigated for content of sulfated glycosaminoglycans (GAGs) in muscle tissue by use of in vivo 35S-sulfate labeling combined with different digestions (papain, chondroitinase ABC, keratanase and nitrous acid treatment), DEAE chromatography, SDS-PAGE and histology techniques. The species investigated in this study have different gaping properties. The non-gaping species, spotted wolffish (Anarhichas minor), contained 3-4 times more 35S-sulfated anionic components than the gaping species, Atlantic cod (Gadus morhua). The higher level of sulfation in wolffish was supported by light microscopy studies using Alcian blue staining with different concentrations of MgCl2 as critical electrolyte. Furthermore, the muscular connective tissue in the non-gaping species was dominated by chondroitin sulfate (CS)/dermatan sulfate (DS), whereas the gaping species was more dominated by heparan sulfate (HS). Moreover, structural differences were observed in the junctions between the myofibers, which were more pronounced in the wolffish. The histological studies revealed that the basement membrane area was rich in acidic mucopolysaccharides in both species.
Asunto(s)
Sulfatos de Condroitina/análisis , Dermatán Sulfato/análisis , Gadus morhua/metabolismo , Heparitina Sulfato/análisis , Músculos/química , Perciformes/metabolismo , Animales , Matriz Extracelular/químicaRESUMEN
A color-based segmentation scheme applied to microscopy images of cryosectioned sausages is proposed. The segmentation scheme is capable of segmenting three different levels on the microscopy images: the fat particles, the protein matrix, and water/starch. The method is based on principal component analysis. A user-friendly program was developed for the manual segmentation of a selection of image pixels by microscopists. Principal component models based on the manually classified pixels are then used to segment fat, protein matrix, and starch/water on microscopy images. The program can also be used as a training tool for microscopists.
Asunto(s)
Análisis de los Alimentos/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Productos de la Carne/análisis , Microscopía/métodos , Análisis de Componente Principal , Animales , Color , Crioultramicrotomía , Grasas/análisis , Microscopía/instrumentación , Proteínas/análisis , Programas Informáticos , Almidón/análisis , Porcinos , Agua/análisisRESUMEN
UNLABELLED: New freezing methods developed with the purpose of improved product quality after thawing can sometimes be difficult to get accepted in the market. The reason for this is the formation of ice crystals that can give the product a temporary color loss and make it less appealing. We have here used microscopy to study ice crystal size as a function of freezing temperature by investigating the voids in the cell tissue left by the ice crystals. We have also investigated how freezing temperature affects the color and the visible absorption spectra of frozen salmon. Freezing temperatures previously determined to be the best for quality after thawing (-40 to -60 °C) were found to cause a substantial loss in perceived color intensity during frozen state. This illustrated the conflict between optimal freezing temperatures with respect to quality after thawing against visual appearance during frozen state. Low freezing temperatures gave many small ice crystals, increased light scattering and an increased absorption level for all wavelengths in the visible region. Increased astaxanthin concentration on the other hand would give higher absorption at 490 nm. The results showed a clear potential of using visible interactance spectroscopy to differentiate between poor product coloration due to lack of pigmentation and temporary color loss due to light scattering by ice crystal. This type of measurements could be a useful tool in the development of new freezing methods and to monitor ice crystal growth during frozen storage. It could also potentially be used by the industry to prove good product quality. PRACTICAL APPLICATION: In this article we have shown that freezing food products at intermediate to low temperatures (-40 to -80 °C) can result in paler color during frozen state, which could affect consumer acceptance. We have also presented a spectroscopic method that can separate between poor product color and temporary color loss due to freezing.